The present invention relates generally to genetic engineering of eukaryotic cellular or viral genomes at specific sites.
A focus of genetic engineering in the recent past has been the use of recombinant DNA methodologies for the purification and amplification of genetic material. U.S. Pat. No. 4,237,224 to Cohen, et al., for example, relates to transformation of procaryotic unicellular host organisms with "hybrid" viral or circular plasmid DNA which includes exogenous DNA sequences. The procedures of the Cohen, et al. patent first involve manufacture of a transformation vector by enzymatically cleaving viral or circular plasmid DNA to form linear DNA strands. Selected foreign DNA strands are also prepared in linear form through use of similar enzymes. The linear viral or plasmid DNA is incubated with the foreign DNA in the presence of ligating enzymes capable of effecting a restoration process, and "hybrids" are formed which include the selected foreign DNA segment "spliced" into the viral or circular DNA plasmid. Transformation of compatible unicellular host organisms with the hybrid vector results in the formation of multiple copies of the foreign DNA in the host cell population. In some instances, the desired result is simply the amplification of the foreign DNA and the "product" harvested is DNA. More frequently, the goal of transformation is the expression by the host cells of the foreign DNA in the form of large scale synthesis of isolatable quantities of commercially significant protein or polypeptide fragments coded for by the foreign DNA.
The success of procedures such as described by Cohen, et al. is due in large part to the ready availability of restriction endonuclease enzymes which facilitate the site-specific cleavage of both the unhybridized DNA vector and, e.g., eukaryotic DNA strands containing the foreign sequences of interest. Cleavage in a manner providing for the formation of complimentary "ends" on the linear DNA strands greatly enhances the likelihood of functional incorporation of the foreign DNA into the vector upon ligating enzyme treatment. Verification of hybrid formation is facilitated by chromatographic techniques which can, for example, distinguish the hybrid plasmids from non-hybrids on the basis of molecular weight. Other useful verification techniques involve radioactive DNA hybridization.
Another manipulative "tool" largely responsible for successes in transformation of procaryotic cells is the use of selectable "marker" gene sequences. Briefly put, hybrid vectors are employed which contain, in addition to the desired foreign DNA, one or more DNA sequences which code for expression of a phenotypic trait capable of distinguishing transformed from non-transformed host cells. Typical marker gene sequences are those which allow a transformed procaryotic cell to survive and propagate in a culture medium containing metals, antibiotics, and like components which would kill or severely inhibit propagation of nontransformed host cells.
In vivo recombination of homologous DNA sequences has been a powerful tool in systems where selections exist for the recombination event. Standard techniques of bacterial genetics rely on recombination of exogenous DNA with homologous DNA on the bacterial chromosome. See, e.g. Miller, Experiments In Molecular Genetics, Coldspring Harbor Laboratory (1972). Recent studies involving introduction of DNA into yeast cells have also shown that recombination of the introduced DNA occurs at homologous sites in the yeast genome. See, e.g., Szostak, et al., Plasmid, 2, pp. 536-554 (1979) and Scherer, et al., Science, 209, 1380-1384 (1980).
Another major focus of genetic engineering has been the manipulation of eukaryotic cell and viral genomes for purposes of attempting correction of genetic defects and modifying antigenicity and pathogenicity of viruses. Such manipulations are significantly more difficult than those involved in the above-noted Cohen, et al. host/vector methodology owing to the larger size and greater complexity of the genomes involved. While a typical DNA plasmid (e.g., Escherichia coli plasmid pBR322) contains about 5,000 nucleotides, the genomes of pathogenic viruses such as herpes virus, pseudorabies and bovine rhinotracheitis virus contain upwards of 150,000 nucleotides. Eukaryotic cell genomes are larger still, involve diploid associations, and are very likely to include multiple alleles of genes of interest.
Site-specific restriction endonuclease enzymes which so greatly facilitate manipulations on small plasmids and bacterial phage DNA are often useless for manipulative work on larger genomes owing to the proliferation of "target" cleavage sites therein. Cleavage of large genomes can be accomplished with relative ease but the existence of multiple cleavage sites renders virtually impossible the properly sequenced reassembly of the genome with ligating enzymes. Thus, while large genomes can readily be fragmented and "mapped" using restriction endonucleases, the enzymatic tools necessary for single, site-specific insertions and deletions are simply not available.
In a like manner, marker gene sequences commonly employed in verification of transformation of procaryotic cell lines are of little use in monitoring for specific insertions and deletions in more complex eukaryotic cell and viral genomes. To be effective in the verification of insertions and/or deletions in such large genomes, marker genes must be susceptible to use in very powerful selection procedures for both the presence and absence of the gene in a transformant genome. They should also be readily obtained and amplified, and should preferably have a relatively small size for convenience in manipulation.
Thus, despite the extensive need for manipulation of eukaryotic cell and viral genomes at specific sites and despite the relative ease of identifying specific DNA sequences which might advantageously be inserted into or deleted from such genomes, the art is without access to procedures which will permit such manipulations and the formation of specifically engineered genomes.
Pertinent to the background of the invention is the disclosure of Pellicer, et al., Science, 209, pp. 1414-1422 (1980) that DNA obtained from viruses and eukaryotic cells has been used to transfer genes coding for growth transformation enzyme, thymidine kinase (tk), adenine phosphoribosyltransferase (APRT) and hypoxanthine-guanine phosphoribosyltransferase (HGPRT) to mutant eukaryotic cell populations deficient for such functions. [See also, Perucho, et al., Nature, 285, pp. 207-210 (1980) for discussion of transfers involving cellular thymidine kinase (i.e., chicken tk) and Lowy, et al., Cell, 22, pp. 817-823 (1980) for discussion of hamster APRT gene characteristics.]
Although Pellicer, et al. report transformation of thymidine kinase deficient (tk.sup.-) mouse fibroblast cells to at least transitorily incorporate herpesvirus tk genes derived from Herpes Simplex Type 1 virus (HSV-l), such transformations have been non-specific as to the site of gene insertion. Due in part to the non-specific nature of insertion, none of the genetic transformations has been or could be purposefully "reversed" by deletion of the inserted tk gene and corresponding reversion of the genome to its initial tk.sup.- state.
The disclosures of applicants and their co-workers appearing in Mocarski, et al., Cell, 22, pp. 243-245 (November, 1980), Post, et al., Cell, 24, pp. 555-565 (May, 1981) and Post, et al., Cell, 25, pp. 227-232 (July, 1981) provide information pertinent to the background of the invention.